Abstract

Cyclooxygenase-2 (COX-2) is an important pharmacological target with
great promise in the prevention and treatment of colorectal cancer
(CRC). The mechanism underlying COX-2 overexpression in CRC is
unresolved. On the basis of the coincident high levels of the
transcription factor c-MYB and COX-2 in CRC, we hypothesized that c-MYB
is a candidate activator of COX-2 transcription. We
identified 13 c-Myb binding sites in the human COX-2
promoter. Eight of these sites were moderate to high-affinity DNA
binding targets. Promoter studies indicated that c-Myb can activate
COX-2 transcription, whereas dominant-negative Myb
mediated repression. These data provide the first rational basis for
overexpression of COX-2 in CRC and offer an additional potential target
for managing this disease.

Introduction

COX-2
3
has been implicated in CRC progression and metastases. Most studies
show that the long-term treatment of individuals with aspirin or
NSAIDS, which target COX-2 activity, reduces colon cancer risk.
Similarly, CRC family cohort members treated with NSAIDS have reduced
CRC incidence
(1)
. In addition, experimental mouse models
using COX-2 knock-out strains crossed with gastrointestinal
cancer prone Min (adenomatous polyposis coli mutant) mice
show a marked reduction in polyp formation and tumor burden
(2)
. Finally, exogenous overexpression of COX-2
accelerates colon cancer in mice
(3)
. c-MYB
overexpression is also a consistent feature of colon tumors and
tumor-derived cell lines
(4)
and is required for cell
growth
(5, 6)
. We have identified an unprecedented number
of c-MYB binding sites in the COX-2 promoter and shown that
c-MYB can efficiently transactivate COX-2 transcription. In
addition, CRC cell lines express both c-MYB and
COX-2 mRNA and coincident high expression of
COX-2 and c-MYB mRNA by in situ
hybridization on colon cancer tissue arrays. These data suggest that
c-MYB is responsible for the elevated levels of COX-2 in colon
epithelial neoplasia and may in itself constitute a possible
therapeutic target for this malignancy.

Materials and Methods

In Situ Hybridization Probes.

c-MYBH riboprobe was generated by insertion
of a cDNA corresponding to the HindIII to BglII
(nucleotides 664–1516) sites encompassing c-MYB
unique sequences that do not cross-react with other MYB
family members. The COX-2 riboprobe (pCRII-huCOX2) was a
gift from Dr. Raymond Du Bois incorporating a 278-bp fragment
corresponding to nucleotides 952-1220.

Hybridization Protocol.

Paraffin-embedded CRC specimens were identified, and core samples were
punched from the block and pooled into arrays for re-embedding. These
were then resectioned to deliver multiple CRC specimens on the same
slide. Slides were incubated in prehybridization solution [60%
deionized formamide, 25 mm Tris (pH 7.4), 1 mm
EDTA (pH 8), 375 mm NaCl, 12% (w/v) dextran sulfate, and
1.2 × Denhart’s solution] for 2 h at 42°C.
Digoxigenin-UTP-labeled RNA at 200 ng/ml in hybridization buffer was
incubated in hybridization buffer with 0.1 mm DTT, 0.1%
sodium thiosulfate, and 0.1% SDS under slide covers overnight at
42°C. Slides were then washed twice in 2× SSC 15 min at room
temperature and then at 42°C, followed by 15-min washes in 1× SSC
and 0.1× SSC. Section were blocked for 30 min at room temperature in
1% Blocking reagent, followed by anti-digoxigenin antibody conjugated
with alkaline phosphatase 1:1000 in 1× blocking solution 60 min at
room temperature, and washed with two buffer changes (0.1 m
maleic acid and 150 mm NaCl) for 15 min each. Color was
developed as described by the manufacturer (Roche). Finally, slides
were rinsed in water and counterstained with 0.1% methyl green, rinsed
in water again, and mounted with Kaiser’s glycerol gelatin.

EMSAs.

Specific DNA binding by CTL c-MYB protein has been described in detail
elsewhere
(7)
. Briefly, double-stranded end-filled
radiolabeled oligonucleotides corresponding to putative c-MYB binding
sites were incubated in the presence of
Baculovirus-expressed recombinant c-MYB for 15 min prior to
running on 6% acrylamide, 0.5× Tris borate EDTA gels. At least
three replicate experiments were performed with 2, 1, and 0.5 ng of
oligonucleotide. Relative binding was quantified using a Molecular
Dynamics PhosphorImager. The oligonucleotide sequences for the
COX-2 promoter used in this study are listed in Table 1
⇓
. All oligonucleotide pairs had 5′-gatc overhangs to
allow labeling.

Hierarchy of c-Myb binding sites in the COX-2 promoter determined by
EMSA

Cloning of the COX-2 Promoter Region.

The COX-2 promoter was amplified as two subclones designated
as COX-2 5′ and 3′ fragments. The 5′ fragment
oligonucleotide primers corresponding to −2355 to −1255 from the
transcription start site (forward,
5′-agctcatggtacacaatagtcacag-3′) and (reverse,
5′-tgaggtgtgaccatggatcaaagtac-3′); noting the presence of
an internal NcoI restriction site used for subsequent
cloning purposes. The 3′ fragment oligonucleotide primers −1255 to +35
(forward, 5-gtactttgatccatggtcacaactca-3′) and (reverse,
5′-gagctgaaggaggcgctgctgaggag-3′). Touchdown PCRs were carried
out at 55°C for 40 cycles on a Perkin-Elmer Thermocycler using Taq
polymerase and Taq start antibody (Bresatec). Fragments corresponding
to the 5′ plus 3′ promoter regions were either joined via an internal
NcoI site or the 3′ fragment alone was subcloned
into pCATBasic (Promega).

CAT Assays.

Fugene 6 (Boehringer Mannheim) transfection of Colo201 and 293HEK cells
were performed according to the manufacturer’s instructions. Cells
were harvested and lysed, adjusted for total protein, and tested forβ
-galactosidase activity
(8)
. Ten μl of protein
extract (adjusted for β-galactosidase activity), 50 μl of 0.25
m Tris (pH 8), 10 μl of acetyl-CoA, and 2 μl of[
14C]chloramphenicol (Amersham) were incubated
for 2–4 h at 37°C. Reactions were extracted in 0.5 ml of ethyl
acetate and dried. The dehydrated pellet was resuspended in 15 μl of
ethyl acetate and was “spotted” on to a TLC plate and
chromatographed in 5% methanol and 95% chloroform. The plate was air
dried, wrapped in plastic, and placed in a PhosphorImager (Molecular
Dynamics).

Expression Plasmids.

Full-length murine c-MYB cDNA was expressed from a
pACT vector described elsewhere
(9)
and the DN
expression construct containing a c-MYB DNA binding domain
fused to the Drosophila engrailed repressor protein and
estrogen receptor (pMERT; Ref.
10
). Modulation
of the DN-Myb construct was achieved using 10−6m 4-OHT (Sigma) added immediately after the
transfection mix was replaced with fresh medium.

Results and Discussion

c-MYB and COX-2 overexpression is a consistent feature of colon
tumors and tumor-derived cell lines; however, they have not been
examined contemporaneously. Therefore, we examined the relationship
between c-MYB and COX-2 expression in CRC
COX-2, c-MYB, and
β2-microglobulin. Five of five
CRC cell lines (Colo201, LIM1215, LIM1863, LIM2405, and LIM2412) had
detectable c-MYB and COX-2 expression, but the
epithelial cell line HEK293 did not (data not shown). It was notable
that Colo201 cells that have an amplified c-MYB locus with
high c-MYB transcription and protein level also had the
highest COX-2 expression (see below).

Similarly, a retrospective survey of the literature is consistent with
the view that c-MYB and COX-2 are overexpressed
in the majority of CRCs; however, an examination of the expression of
both of these genes in the same primary tumor samples has not been
performed. Hence, we investigated 20 CRC specimens from the Peter
MacCallum Cancer Institute archives by in situ hybridization
using antisense RNA encoded by c-MYB and COX-2
cDNAs. These specimens were chosen on the basis of clear histological
features of CRC. Fig. 1A⇓
shows high expression of both COX-2 and
c-MYB mRNA in the epithelial components of two
representative CRC biopsy sections examined as indicated by the purple
staining (D2 and E3). These images are taken at low power magnification
(×25) to show the distribution of mRNA within a field of tumor tissue
and were consistent for 20 matched core samples. Sense and antisense
probe hybridization for either c-MYB or COX-2
conducted on consecutive sections of the two independent core
samples examined at higher power magnification (×100) shows the
colocalization of COX-2 and c-MYB to the
epithelial cells in these tumors (Fig. 1B⇓
). In general, this
analysis confirmed the restricted expression of COX-2 and
c-MYB to the epithelial cells and low expression in stroma
and muscle. As expected, specific c-MYB expression was
evident in the normal epithelial mucosa confined to the columnar cells
of the colonic crypt (Ref.
4
; Fig. 1C⇓
).

In situ hybridization of tissue arrays
generated from fully characterized paraffin-embedded CRC specimens.
Sections were hybridized with either sense or antisense probes
corresponding to COX-2 and c-MYB cDNA.
A, hybridization of sense and antisense probes to core
D2 and E3 cut at different planes within the same blocks (×25).
B, hybridization of consecutive sections of the same
biopsy specimens showing colocalized hybridization of
c-MYB and COX-2 (×100).
C, hybridization of specimens showing restricted
expression of c-MYB and COX-2 to the
columnar cells of colonic crypts (arrows; ×630).
Notably, this level of expression was considerably lower than that
observed in adjacent tumor samples.

Having established the colocalization and expression of
c-MYB and COX-2, we next considered the
possibility that c-MYB may act upon the COX-2 promoter.
Examination of the human cox-2 gene in the 5′ region
upstream of the first exon
(11)
revealed 13 putative c-MYB
binding sites that conform to the core binding consensus c/tAACt/g
(Ref.
12
; Fig. 2
⇓
). Analysis of the human COX-1 promoter region
(13)
did not detect any such sequences. To examine whether
these putative sites can be recognized by c-MYB protein,
double-stranded oligonucleotide duplexes corresponding to these sites
sequentially identified as sites “A” through “M” were
synthesized. These were radiolabeled by end-filling reaction in the
presence of [α-P32]dATP, incubated in the
presence of recombinant c-MYB, and subjected to EMSA
(7)
.

Sequence analysis of the human COX-2
promoter region highlighting potential c-MYB binding sites (c/tAACt/g)
as boxes with their alphabetical designation noted in
the left-hand margin from A to M plus site R identified
in the rat COX-2 promoter region. The TATA box and
transcription start site (arrow) are also indicated.
Sites L and M are in the reverse orientation. Boxes with
underlined sequences are predicted to be high-affinity
sites because of the presence of a guanine residue at position 6. The
location of the NcoI restriction site is noted as the
joining point for the 5′ and 3′ segments of the COX-2
promoter sequence.

Each oligonucleotide pair was tested in comparison to at least
three different pairs corresponding to other putative sites to assemble
a hierarchy of relative binding affinities. An additional site called“
R” was examined because it was the only perfectly aligned (but not
sequence-conserved) site present in a comparison of the rat
(14)
and human COX-2 5′ sequences (see below).
Three oligonucleotide concentrations were used to allow an estimate of
binding, to achieve linear binding, and finally to detect minimal
binding of c-MYB to the lowest affinity sites. All EMSA reactions were
controlled for nonspecific DNA binding and used the same batch of CTL
recombinant c-MYB. Table 1
⇓
lists the sites in order of binding
affinity, whereby sites L, C, D, F, and I belong to the high-affinity
group; sites K, E, and H have intermediate relative affinity, and sites
B, G, and A fall into the low-affinity group. Sites J, M, and R serve
as poor c-MYB binding sites. From studies of the specific DNA binding
of c-MYB
(12)
, it is clear that flanking nucleotides
adjacent to the core consensus sequence (pyAACt/g) influence
high-affinity binding. Of utmost importance to high-affinity binding is
a nucleotide residue position 6 that is ideally a guanine. The
high-affinity sites L, C, D, F, and I conform to this sequence
requirement as does site K, which is the next best bound site that has
been assigned to the moderate-affinity group.

Putative c-MYB target genes have been reported elsewhere
(15)
; however, the presence of 13 theoretical and 8
high-affinity c-MYB binding sites within the promoter of a gene is
unprecedented. The absence of putative c-MYB sites within a similar
nucleotide stretch in the promoter region of the COX-1 gene
that encodes a highly related isoform of COX-2 reinforces the
nonrandomness of this observation.

To determine whether the COX-2 promoter could be
transactivated by c-MYB, we cloned the 5′ region of the human
COX-2 gene in two parts, subcloned them collectively or as
the most proximal (3′) part of the promoter region, into the
pCATBasic vector and conducted transactivation studies. A
histogram depicted in Fig. 3A⇓
indicates that the most proximal region of the
COX-2 promoter (3′) is very active in the colon cell
line Colo201 (∼8-fold activation) compared with the chicken β-actin
promoter (2.5-fold) driving the CAT reporter gene. Notably,
Colo201 expresses the highest level of endogenous COX-2 mRNA
and c-MYB.

Transactivation of the COX-2 promoter in
c-Myb-expressing colon cell line Colo201 and in c-Myb-negative cell
line HEK293 by exogenous c-MYB. A, the 3′ cox-2 promoter
region results in ∼8-fold activation of the CAT
reporter gene in Colo201 cells compared with the β-actin-driven CAT
construct (pACTCAT). This cell line expresses relatively
high c-MYB levels. B, in c-MYB-negative HEK293 cells,
the addition of exogenous c-MYB transactivates the 3′ region of the
COX-2 reporter construct 4-fold, whereas the addition of
a DN-Myb construct has no effect unless activated by the addition of
4-OHT. C, the combined 5′ and 3′ regions of the
COX-2 promoter allows slightly higher transactivation
compared with the 3′ region alone. In addition, the DN construct also
inhibits its activity. Bars, SE.

To further characterize the cox-2 promoter, we transfected
the 3′ and the 5′ plus 3′ COX-2 promoter regions into the
human epithelial cell line HEK293, which has undetectable
c-MYB and COX-2 mRNA. Fig. 3B⇓
shows
that full-length c-MYB can transactivate the 3′ cox-2
promoter 4-fold compared with the basal level of transcription observed
in HEK293 cells, whereas the combined 5′ + 3′ promoter
fragment allows ∼5-fold transactivation. Exogenous c-MYB expression
in the HEK293 cells was confirmed by Western blotting (data not shown).

Additional evidence that the c-MYB binding sites in
COX-2 are important is demonstrated when transactivation of
either the 5′ + 3′ or 3′ promoter constructs are examined in
the presence of a DN (DN-Myb) form of c-MYB. The activity of this
construct is dependent upon the addition of 4-OHT. DN-MYB plus 4-OHT
substantially reduced the transactivation of both COX-2
reporter constructs. 4-OHT alone did not affect full-length
c-MYB-dependent transactivation (data not shown). These data show that
c-MYB can activate the COX-2 promoter. They further show
that the DN-MYB construct inhibits the basal level of transcription
most likely by the direct binding to, and repression through, the
numerous c-MYB binding sites characterized in the EMSA studies
documented above.

An analysis of potential c-MYB binding sites within the rat
(14)
and mouse
(16)COX-2
promoters revealed 10 and 13 probable sites, respectively, with 4 in
each of these satisfying the criteria for high-affinity binding sites.
The distribution of probable high-affinity c-MYB binding sites within
the mammalian COX-2 promoters is similar, and sequence
comparisons show 85% similarity between the rat and mouse and 69%
similarity between the human and mouse. However, the sites do not align
perfectly with each other, suggesting that substantial evolutionary
divergence has occurred between the three mammalian species.
Nevertheless, these observations are consistent with COX-2
regulation by c-MYB during colon tumorigenesis in rodents. Further
support of these views is the observation of overexpression of c-MYB in
rat colon tumors
(17)
.

The data presented here suggest that the high level of COX-2
mRNA associated with colon tumor epithelial cells may be attributable
to the transcriptional activation of the COX-2 promoter by
c-MYB. Perhaps because of the historical association of c-MYB and
hematopoiesis, the role of c-MYB in other cell types has been largely
ignored. Nevertheless, we have shown that c-MYB appears to be an
important regulator of BCL-2 and thus apoptosis in developing murine
colon and in human colon tumor cell lines
(4, 18)
. The
observations presented here highlight an additional connection between
c-MYB and the inhibition of apoptosis because COX-2 overexpression also
appears to be protective against apoptosis
(19)
. Thus,
c-MYB overexpression in CRC may inhibit apoptosis by direct regulation
of both BCL-2 and COX-2.

COX-2 appears to inhibit apoptosis, and it is likely that therapeutic
targeting of COX-2 activity in CRC prevention and treatment may restore
normal apoptosis. We have also suggested that c-MYB regulates apoptosis
in normal and malignant colon tissues through the transcriptional
control of BCL-2. The ablation of c-MYB
expression in tumors may lead to the reduction of downstream
transcriptional targets. To this end, successful inhibition of
c-MYB expression has been achieved in vivo using
antisense oligonucleotide treatment
(20)
, and these
methodologies have been used to inhibit malignant cell growth and
reduced tumor burden. These end points have been achieved with minimal
apparent effects on tissues that normally express c-MYB.
These kinds of studies are the basis of Phase II clinical trials
(National Cancer Institute-sponsored Protocol Ids UPCC-3492 and
NCI-H94–0532). These data offer encouraging prospects for targeting
CRC with elevated COX-2 and BCL-2 expression
through the inhibition of c-MYB expression.

Acknowledgments

We thank Dr. Kathy Weston for generous provision of reagents and
advice. We also thank Drs. Maree Overall and Grant McArthur for
critical reading of the manuscript and Assoc. Prof. David
Bowtell and Prof. Joe Sambrook for constructive comments.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported by a National Health and
Medical Research Council Research Fellowship and the Anti-Cancer
Council of Victoria, Australia.